Obesity and its associated diseases, type 2 diabetes, heart disease, and cancer have become a leading cause of death worldwide. Unfortunately, only risky surgical procedures constitute an effective long-term treatment. Better understanding of the molecular mechanisms underlying obesity is necessary for the development of urgently needed therapeutics. Important genetic regulators of human metabolism, including key components of the insulin signaling pathway, were first discovered in the roundworm Caenorhabditis elegans. Despite the great potential of C. elegans for gene and drug discovery through automated high-throughput (HT) screens, the required methodology has not yet been developed. To fill this void, I have been developing technology that will allow automated HT C. elegans-based RNAi screens (O'Rourke et al, 2009). At the same time, in the course of studying the C. elegans response to fasting, I uncovered that the most commonly used assay to assess fat accumulation in C. elegans, Nile Red staining, only stains a small subset of fats contained in Iysosomes. I therefore developed an alternative methodology that reads out C. elegans major fat stores, Oil-Red-O staining. Using Oil-Red-O staining in combination with the HT methodology, I am proposing to perform an automated RNAi screen for genes that regulate fat accumulation in C. elegans. As previous screens used Nile Red as their readout, the proposed screen will constitute the first screen for genes that regulate major fat stores in a whole-living animal. In parallel, I discovered that there is transcriptional activation of lipolysis (fat breakdown) that occurs when either C. elegans and [sic] mice are fasted, leading, in the case of C. elegans, to a 30% decrease in fat stores (O'Rourke et al, submitted to Science). In worms, this transcriptional activation of lipolysis is mediated by the inactivation of the transcriptional repressor mx/-3. In turn, preliminary data suggest that mx/-3 inactivation is mediated by small RNAs. The role of small RNAs in the control of energy balance is largely unexplored. In order to explore the role of small RNAs in energy homeostasis I propose to sequence all small RNAs from fasted and well-fed worms. Finishing the optimization of the HT screening methodology will allow me to acquire knowledge in automated image analysis and large-scale data processing. The screen for genes that regulate fat accumulation will provide me with experience in functional genomics. By mining and characterizing small RNAs, I will familiarize with cutting-edge deep sequencing technologies. The knowledge, reagents, publications, and collaborations gained and generated during this training period will greatly facilitate my transition to a successful independent career in the field of metabolism. The completion of the development of the proposed HT screening methodology not only constitutes an enabling technology for the whole C. elegans community, but it will initially provide me with a unique advantage as an independent researcher. The pilot gene search performed during the mentored phase will constitute a proof-of-principle to be exploited during the independent phase of the award when large-scale screens for genes and pilot screens for drugs that alter fat accumulation will be performed. Also during the independent phase, I will perform an in depth genetic and biochemical analysis of the genes and small RNA regulators of metabolism uncovered during the mentored phase. I anticipate that my research will uncover molecules and biological pathways that regulate energy balance. This will accelerate the design and testing of drugs to prevent and treat the major human health problem of obesity. PUBLIC HEALTH RELEVANCE: Obesity has become a leading cause of death worldwide. Genetic components, as well as the environment contribute to the severity of the syndrome. Caenorhabditis elegans is a simple model organism, a roundworm, sharing 50% of their genes with humans. C. elegans has been successfully used to uncover genes and drugs relevant to human disease in the past. I am going to work on developing an enabling technology that will accelerate the discovery of genes that could predispose and compounds that could relief human disease. I will particularly focus on finding genes that control fat accumulation and compounds that promote lipolysis (the process of breaking down fats). Learning more about the molecular mechanisms that lead to the activation of lipolysis could allow us to manipulate the balance between fat storage and consumption. Drugs that activate the breakdown of lipids could be used as therapeutic agents for the treatment of obesity.